This application is a continuation 371 of PCT/EP08/55535 filed May 6, 2008 which claims priority to German 10 2007 022 3678, filed May 7, 2007.
The present invention concerns an in vitro method for medical diagnosis, prognosis and therapy follow-up for patients having a cardiac disease or being suspected of developing or having a cardiac disease comprising the steps of:                providing a sample of a patient having a cardiac disease or being suspected of developing or having a cardiac disease,        determining amino-terminal proANP or partial peptides thereof having from 12 to 98 amino acids in said sample using at least one antibody that binds specifically to a partial sequence of amino-terminal proANP,        attributing the determined amino-terminal proANP level or the level of partial peptides thereof to a clinical picture wherein the attribution is carried out independent of the BMI of the patient.        
The present invention further concerns a rapid test assay for conducting the method of the present invention and the use of antibodies suitable for the method and assays according to the present invention.
Heart failure (HF) is common, associated with high morbidity and mortality, and difficult to diagnose, particularly in the emergency department (ED) [Cleland, J. G. et al., Eur Heart J, 2003. 24(5): p. 442-63; Mosterd, A. et al., Heart, 2007. 93(9): p. 1137-46]. Dyspnea is the leading symptom of most HF patients. Unfortunately, neither patient history nor physical examination can accurately differentiate dyspnea due to HF from dyspnea due to other causes, such as pulmonary diseases [Mueller, C. et al., Can J Cardiol, 2005. 21(11): p. 921-4; Wang, C. S. et al., Jama, 2005. 294(15): p. 1944-56]. However, accurate diagnosis is mandatory for the selection of the most appropriate treatment.
B-type natriuretic peptides are quantitative markers of HF that have been shown to be very helpful in the diagnosis of HF. The use of B-type natriuretic peptide (BNP) and its amino-terminal fragment, N-terminal pro-B-type natriuretic peptide (NT-proBNP) significantly increases the diagnostic accuracy in the ED [Januzzi, J. L., Jr., et al., Am J Cardiol, 2005. 95(8): p. 948-54; Maisel, A. S. et al., N Engl J Med, 2002. 347(3): p. 161-7], and thereby improves patient evaluation and treatment [Moe, G. W. et al., Circulation, 2007. 115(24): p. 3103-10; Mueller, C. et al., N Engl J Med, 2004. 350(7): p. 647-54].
The concentration of atrial natriuretic peptide (ANP) in the circulation is approximately 50- to 100- fold higher than BNP [Pandey, K. N., Peptides, 2005. 26(6): p. 901-32.]. Therefore, the biological signal reflected by the increased ANP may be pathophysiologically and therefore diagnostically even more important than the signal of BNP. Despite this, little is known about the diagnostic performance of ANP and its precursors [Cowie, M. R. et al., Lancet, 1997. 350(9088): p. 1349-53]. Mature ANP is derived from the precursor N-terminal-proANP(NT-proANP), which is significantly more stable in the circulation than the mature peptide and is therefore thought to be a more reliable analyte [Vesely, D. L., IUBMB Life, 2002. 53(3): p. 153-9]. Nevertheless, due to the fact that NT-proANP might be subject to further fragmentation [Cappellin, E. et al., Clin Chim Acta, 2001. 310(1): p. 49-52], immunoassays for measurement of mid-regional proANP (MR-proANP) may have an advantage [Morgenthaler, N. G. et al., Clin Chem, 2004. 50(1): p. 234-6].
Cardiac diseases, such as chronic heart failure (CHF), acute heart failure, acute coronary syndrome and myocardial infarction are widely spread and often have a devastating prognosis. Early prognosis of the cardiac disease can improve patient outcome through timely preventive and therapeutic measures [Jortani, S. A. et al., Clin Chem 2004; 50:265-278]. A wealth of data suggest that the assessment of natriuretic peptides represents a useful addition to chest X-ray, electrocardiogram, and doppler echocardigraphy in verifying the clinical diagnosis and prognosis for suspected cardiac diseases [Cowie, M. R. et al., Eur Heart J 2003; 24:1710-1718]. The most important members of the family of natriuretic peptides are atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Both are markers of cardiac function. In addition to the use in helping to establish the diagnosis of CHF [Cowie, M. R. et al., Lancet, 1997. 350(9088): p. 1349-53; Mueller, C. et al., N Engl J Med, 2004. 350(7): p. 647-54], they are used in the risk stratification of patients with stable or recently decompensated CHF [Omland, T. et al., Cirulation 1996; 93:1963-1969; Anand, I. S. et al., Circulation 2003; 107:1278-1283; Cheng, V. et al, J Am Coll Cardiol 2001, 37:386-391], or with acute coronary syndrome [Morrow, D. A. et al., JAMA 2005, December 14; 294(22):2866-71] and, in addition, are used in follow-up and guidance in CHF therapy [Troughton, R. W. et al., Lancet 2000, 355:1126-1130].
Both BNP and the other cleavage product of its precursor, N-terminal proBNP(NT-proBNP) are currently used in the assessment of patients with CHF. ANP originates from the cleavage of its precursor proANP. The other cleavage product, N-terminal proANP (NT-proANP) is significantly more stable in circulation than ANP. Therefore, NT-proANP has been suggested to be a more reliable analyte in the prior art [Ala-Kopsala, M. et al., Clin Chem, 2004. 50(9): p. 1576-88]. However, the N- and C-terminal regions of the N-terminal pro-peptide can be altered through enzymatic degradation [Seidler, T. et al., Biochem Biophys Res Commun 1999, 255:495-501]. Commonly, BNP and NT-proBNP are considered as being more suitable for the diagnosis and prognosis in patients with cardiac insufficiency than ANP and NT-proANP. The reason for this may be that the determination of ANP and NT-proANP is considered less reproducible, and that conceptual flaws in most of the applied assay designs lead to a partial underestimation of the amount of circulating analyte [Yoshibayashi, M. et al., Eur J. Endocrinol 1996; 135:265-268; Vesely, D. L. et al., IUBMB Life, 2002. 53(3): p. 153-9].
Usually, a peptide comprising 28 amino acids (99-126) of the C-terminal section of a pro-hormone comprising 128 amino acids (proANP; SEQ ID NO: 2) is referred to as the actual hormone ANP. Upon release of ANP from its pro-hormone proANP, an equimolar amount of the remaining larger partial peptide of proANP, the N-terminal proANP, consisting of 98 amino acids (NT-proANP; proANP (1-98)) is released into circulation. As NT-proANP possesses a significantly greater half life time and stability NT-proANP can be used as laboratory parameter for diagnosis, follow-up and therapy control in cardiac diseases. For a deeper insight into the topic please refer to: Lothar Thomas (Editor), Labor and Diagnose, 5th expanded ed., sub-chapter 2.14 of chapter 2, Kardiale Diagnostik, pages 116-118, and the literature cited therein.
Both for the determination of ANP itself as well as for the determination of NT-proANP in biological liquids (serum, plasma, urine), various immunoassays have been developed in the past and have been applied in clinical research and practice. The major part of such immunoassays for the determination of ANP and NT-proANP is based on the known principle of competitive immunoassays, the best known of which is the radioimmunoassay (RIA). Competitive immunoassays for the determination of proANP and ANP are described or used, for instance in [Cappellin, E. et al., Clin Chim Acta, 2001. 310(1): p. 49-52] and [Hiroshi Itoh et al., Journal of Hypertension 1988, 6 (suppl 4) S309-S319; Meleagros, L. et al., Peptides, Vol. 10, 545-550, 1989; Amir Lerman et al., Lancet 1993, 341:1105-09, 1993; John G F Cleland et al., Heart 1996, 75:410-413; Martin G. Buckley et al., Clinical Science (1999) 97:689-695].
By the use of various antibodies which recognize various sequences of NT-proANP (NT-proANP 1-98; SEQ ID NO: 1) for the determination of NT-proANP with competitive immunoassays, it was interpreted that various peptides appear in biological liquids, in particular blood or urine, which correspond to degradation products of NT-proANP (proANP 1-98). In particular, it has been interpreted that low molecular fragments are formed form NT-proANP, in particular such fragments, to which the amino acid sequences 1-30, 31-67 and 79-98 of proANP have been addressed due to their immunoactivity (cf. e.g. M. G. Buckley et al., Clinica Chimica Acta, 191 (1990) 1-14; J. B. Hansen et al., Lab. Invest. 1995, 55:447-452; Rose M. Overton et al., Human Plasma and Serum; Peptides, Vol. 17, 1155-1162, 1996; Sreedevi Daggubati et al., Cardiovascular Research 36 (1997) 246-255; E. F. Macaulay Hunter et al., Scand J Clin Invest 1998; 58: 205-216; Martina Franz et al., Kidney International, Vol. 58 (2000), 374-383, Fumiaki Marumo et al., Human Plasma and Urine; Biochem. Biophys. Res. Commun. 137:231-236 (1985) and Engelbert Hartter et al., Clin Chem Lab Med 2000, 38(19): 27-32).
Competitive immunoassays which specifically recognize the above-mentioned sequences always recognize the full NT-proANP in the same manner—disregarding influences based on different avidities or possible conformational influences—and do therefore not differentiate between NT-proANP and its fragments.
In contrast to competitive immunoassays, non-competitive sandwich immunoassays show a number of advantages. For instance, they can be interpreted as solid phase assays (heterogeneous assays) more readily, are more robust, provide measurement results with a greater sensitivity, exhibit less cross-reactivity, and are more suitable for automation and serial measurements. Furthermore, by recognizing only such molecules and/or peptides in which both binding moieties for the antibodies which are used for the sandwich formation are present in the same molecule, sandwich immunoassays may lead to slightly different conclusions in comparison with competitive immunoassays which only apply one type of antibody. If, for instance, the binding areas are located on different partial peptides (degradation product, fragments) a binding of the antibodies to such fragments will not lead to the measurement signal which is typical for the complete “sandwich”.
Due to the known advantages of sandwich immunoassays in principle as well as due to the possibility of selectively measuring only complete NT-proANP without any influence of degradation products and fragments, sandwich immunoassays for the determination of NT-proANP have already been described and applied in clinical research and practice. For instance, EP 721 105 B1 describes a sandwich immunoassay for the determination of proANP, in particular in the area of diagnosis of cardiac diseases and chronic kidney failure, to using two monoclonal antibodies, one of which binds to amino acids 1-25 of proANP (compare EP 350 218 B1) and the other of which binds to amino acids 43-66 of proANP. By this kind of sandwich immunoassay, only such proANP partial peptides are detected or co-detected in the presence of proANP (1-98), which comprise at least the first 66 amino acids of the proANP sequence.
Another sandwich immunoassay which is similar in this respect is described in [Mats Stridsberg et al., J. Med. Sci 102, 99-108, Vol. 18: 1201-1203]. This sandwich immunoassay also uses two monoclonal antibodies, one of which binds to amino acids 1-30 of proANP, while the other binds to amino acids 79-98. Due to the choice of binding areas at the terminal ends of proANP (1-98) it can be assumed that this sandwich immunoassay will only detect intact proANP (1-98).
WO 00/19207 describes a method for the determination of proANP (1-98) in which two of three polyclonal antibodies are used, which bind to the amino acid sequences 8-27, 31-64 or 79-98 of the proANP (1-98) sequence. The assay for the determination of proANP (1-98) commercially offered by the applicant of WO 00/19207 is an enzyme immunoassay, using a pair of affinity purified polyclonal sheep antibodies, of which the immobilized antibody recognizes amino acids 10-19 of proANP, while a second polyclonal antibody is used for detection, which recognizes amino acids (85-90) (compare operation manual of the sandwich assay proANP (1-98) by BIOMEDICA, A-1210 Wien). Thus, this assay also recognizes only peptide species containing the terminal ends of the full proANP (1-98), i.e. complete or nearly complete NT-proANP).
All competitive assays or assays which apply the sandwich principle described in the prior art have basically been developed as assays for cardiac diagnostics or which have been used in the context of cardiac diagnostics, whereby chronic kidney failure has been mentioned as further diagnostic application (compare EP 721 105 B1 and citation [Buckley M G et al., AM. J. Hypertens. 1990 December, 3 (12 Pt 1): 933-935]).
Furthermore, a new sandwich immunoassay for the determination of the central region of proANP (amino acids 53-73) has been developed, the measurement results of which are not influenced by a putative terminal truncation of the peptide [Clinical Chemistry 50, No. 1, 2004, pages 234-236].
It was an object of the invention to provide an in vitro method for medical diagnosis, prognosis and therapy follow-up for patients having a cardiac disease or being suspected of developing or having a cardiac disease which provides reliable information especially to the medical practitioner in the Emergency Department (ED).
Thus, the present invention comprises an in vitro method for medical diagnosis, prognosis and therapy follow-up for patients having a cardiac disease or being suspected of developing or having a cardiac disease comprising the steps of:                providing a sample of a patient having a cardiac disease or being suspected of developing or having a cardiac disease,        determining amino-terminal proANP or partial peptides thereof having from 12 to 98 amino acids in said sample using at least one antibody that binds specifically to a partial sequence of amino-terminal proANP,        attributing the determined amino-terminal proANP level or the level of partial peptides thereof to a clinical picture wherein the attribution is carried out independent of the BMI of the patient.        
Preferably, the in vitro method according to the present invention is carried out, wherein the attribution to a clinical picture is carried out with help of a BMI independent cut-off value.
Attribution to a clinical picture means that the determined amino-terminal proANP level or the level of partial peptides thereof is used for the medical diagnosis, prognosis or therapy follow-up for patients having a cardiac disease or being suspected of developing or having a cardiac disease wherein the use is carried out independent of the BMI of the patient.
The cardiac disease of the patient may be selected from but not restricted to chronic heart failure, acute heart failure, acute coronary syndrome or myocardial infarction.
In one embodiment of the invention the disease is diagnosed, thus said method is a method for medical diagnosis.
In one preferred embodiment of the invention the cardiac disease is heart failure. The in vitro method according to the invention is especially well-suited and reliable for patients that exhibit symptoms of dyspnea and are therefore suspected of having heart failure and the clinical picture is diagnosed. The evaluation and management of patients presenting to the Emergency Department is facilitated by the method according to the present invention. Accurate diagnosis is mandatory for the selection of the most appropriate treatment for such patients. Therefore, the present invention meets the need to accurately differentiate dyspnea due to heart failure from dyspnea due to other causes, such as pulmonary diseases. The method according to the present invention, therefore, significantly improves diagnostic accuracy for heart failure.